Various methods may be used in biology and in medicine to detect different targets in a biological sample. For example, analysis of proteins in histological sections and other cytological preparations may be performed using the techniques of histochemistry, immunohistochemistry (IHC), or immunofluorescence. Analysis of proteins in biological samples may also be performed using solid-state immunoassays, for example, using the techniques of western blots, or using cell-based assays that can be performed, for example, by using flow cytometry.
Many of the current techniques may detect only a few targets at one time (such as IHC or fluorescence-based Western blots where number of targets detectable is limited by the fluorescence-based detection system) in a single sample. Further analysis of targets may require use of additional biological samples from the source, limiting the ability to determine relative characteristics of the targets such as the presence, absence, concentration, and/or the spatial distribution of multiple biological targets in the biological sample. Moreover, in certain instances, a limited amount of sample may be available for analysis or the individual sample may require further analysis.
Methods of iteratively analyzing an individual sample are described in U.S. Pat. Nos. 7,629,125 and 7,741,046. In particular, U.S. Pat. No. 7,741,046 provides methods of detecting multiple targets in a biological sample that involve the use of oxidation for inactivating signal generators (e.g., for bleaching fluorescent dyes.) The oxidation reaction is accomplished by using oxidizing reagents, such as hydrogen peroxide.
Additionally, a signal can be inactivated by continuous exposure of the signal generator to irradiation, i.e., by photobleaching. Similar to signal inactivation by oxidation, this process can be lengthy and may not proceed to completion, resulting in reduced signal-to-noise ratio. In addition, continued exposure of sample to irradiation may damage the biological sample. These prior methods also occasionally affect protein epitopes and in such cases either these epitopes have to be detected in the first round or antibodies to alternate epitopes or downstream pathway proteins have to be used to study their effects on disease. In some cases the antigenicity is further enhanced for targets tested in later rounds preventing meaningful comparison of expression.
Photobleaching of cyanine dyes using radicals generated from radical photoinitiators have been recently described. The concept of cyanine dye bleaching using radicals was described, see J Am Chem Soc. 2009 Dec. 30; 131(51): 18192-18193; Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 48, 4243-4251 (2010). Using silver radicals for cyanine dye bleaching was described in J. Am. Chem. Soc., 2001, 123 (33), pp 7985-7995. The direct and sensitized mechanism of triazine-dye component system was described in Chem. Mater. 1997, 9, 1353-1361. However, none of these references discusses the suitability of photobleaching using radical initiators for signal cycling in a multiplexed assay of a biological sample.
Thus, there still remains a need for fast, milder and more sensitive methods for sequential analysis of biological targets.